Incidental prostate cancer prevalence at radical cystoprostatectomy--importance of the histopathological work-up.

The reported incidental prostate cancer prevalence rates at radical cystoprostatectomy cover a range from 4 to 60 %. We investigated the influence of the histopathological work-up on prostate cancer prevalence rates. We identified 114 patients who had undergone cystoprostatectomy for bladder cancer between 2000 and 2012. Complete histopathological assessment was defined as follows: (i) complete embedding of the prostate gland, (ii) sectioning of 15 or more prostate sections, and (iii) processing as whole mount slides. Prostate cancer prevalence rates derived from complete and incomplete histopathological assessments were compared. The overall prostate cancer prevalence rate was 59.6 %. A mean of 14.4 macroscopic tissue sections (thickness 3-5 mm) were sectioned. Sectioning ≥15 sections resulted in a prostate cancer detection rate of 75 %, compared to 42.6 % when sectioning <15 sections (p < 0.001). Complete embedding yielded a prostate cancer detection rate of 72.3 and of 23.1 % for partly embedded prostates (p < 0.0001). Prostate cancer was detected in 68.8 % of the whole mounted samples and in 38.2 % of the samples sectioned as standard slides (p < 0.01); according to the criteria described by Epstein and Ohori, 44.1 % of the detected prostate cancers were clinically significant. The quality of the histopathological work-up significantly influences prostate cancer detection rates and might at least partially explain the highly variable reported incidental prostate cancer prevalence rates at cystoprostatectomy (CP). The high proportion of significant prostate cancer found in our series calls for a careful surgical approach to the prostate during CP.

ERG rearrangement and protein expression in the progression to castration-resistant prostate cancer.

BACKGROUND: Approximately half of the prostate carcinomas are characterized by a chromosomal rearrangement fusing the androgen-regulated gene TMPRSS2 to the oncogenic ETS transcription factor ERG. Aim of this study was to comprehensively analyze the role and impact of the ERG rearrangement and protein expression on the progression to castration-resistant (CR) disease. METHODS: We used a tissue microarray (TMA) constructed from 114 hormone naive (HN) and 117 CR PCs. We analyzed the ERG rearrangement status by fluorescence in situ hybridization and the expression profiles of ERG, androgen receptor (AR) and the proliferation marker Ki67 by immunohistochemistry. RESULTS: Nearly half of the PC tissue specimens (HN: 38%, CR: 46%) harbored a TMPRSS2-ERG gene fusion. HN PCs with positive translocation status showed increased tumor cell proliferation (P<0.05). As expected, TMPRSS2-ERG gene fusion was strongly associated with increased ERG protein expression in HN and CR PCs (both P<0.0001). Remarkably, the study revealed a subgroup (26%) of CR PCs with ERG rearrangement but without any detectable ERG protein expression. This subgroup showed significantly lower levels of AR protein expression and androgen-regulated serum PSA (both P<0.05). CONCLUSIONS: In this study, we identified a subgroup of ERG-rearranged CR PCs without detectable ERG protein expression. Our results suggest that this subgroup could represent CR PCs with a dispensed AR pathway. These tumors might represent a thus far unrecognized subset of patients with AR-independent CR PC who may not benefit from conventional therapy directed against the AR pathway.

[Renal masses and indication to nephrectomy: preoperative assessment and clinical presentation of five cases]. / Masses rénales et indication à la néphrectomie. Clinique et bilan préopératoire de cinq cas.

In Switzerland, renal cancer represents 2% of adult neoplasia and its incidence is 8,2 cases per 100000 people. Small renal masses (< 4 cm) are mostly found incidentally during ultrasound or CT-scan evaluations. Differentiation between benign lesions (20% of renal masses < 4 cm), cancer and pseudotumors may be difficult. CT-scan plays a central role in the preoperative assessment, but the use of renal biopsy seems to have a growing importance in this assessment. This article summarizes the different diagnostic approaches in the preoperative assessment and describes five cases of nephrectomy for renal masses incidentally discovered.

[Multiprobe FISH for enhanced prediction of recurrent bladder cancer]. / FISH für eine verbesserte Abschätzung des rezidivrisikos nach transurethraler Resektion von Urothelkarzinomen.

The early detection of recurrent urothelial cancer is a great challenge for both urologists and pathologists. Cytology is a sensitive and highly specific tool for the diagnosis of high-grade bladder cancer and carcinoma in situ, but is not reliable in low-grade tumors. Therefore, cystoscopy has remained the gold standard for surveillance after resection of bladder cancer. Recent data suggest that multi-target fluorescence in situ hybridization (FISH) can markedly improve the sensitivity of urinary cytology. The goal of this study was to investigate the utility of the recently developed FISH assay Uro-Vysion for the prediction of recurrence during surveillance. 134 bladder washes obtained during a negative follow-up cystoscopy from 127 patients were analyzed. Positive cytology was strongly associated with subsequent recurrence, emphasizing the importance of conventional cytology for bladder cancer surveillance. A positive UroVysion FISH test significantly predicted recurrence when cases with rare tetraploic cells were considered as negative. Taken together, both cytology and FISH help to better determine the risk of bladder cancer recurrence in order to establish more individualized follow-up schemes.

Overexpression of the cytoprotective protein clusterin decreases radiosensitivity in the human LNCaP prostate tumour model.

OBJECTIVE: To evaluate the effect of clusterin overexpression on radiation-induced tumour growth rates and apoptosis in human prostate LNCaP cells, as prostate cancer cells are relatively resistant to radiation-induced apoptosis and local recurrences are common, but overexpression of the anti-apoptotic protein clusterin can accelerate progression to androgen-independence and to confer a chemoresistant phenotype in various prostate cancer models. MATERIALS AND METHODS: Western blot analysis and immunohistochemistry were used to compare clusterin expression levels in parental (P) and clusterin-transfected (T) LNCaP cells in vitro and in vivo. The effects of radiation on clusterin-expression in both parental LNCaP/P and clusterin-transfected LNCaP/T tumours were analysed by Northern blot analysis. The cellular response to radiation was determined up to 3 weeks after irradiation using tetrazolium and re-growth assays, and cell-cycle analysis by flow cytometry. RESULTS: Clusterin mRNA expression increased from undetectable to low levels in LNCaP/P tumours after radiation and more than three-fold in LNCaP/T tumours. Clusterin overexpression decreased the radiosensitivity in a time-dependent manner, reducing the extent of growth arrest and apoptosis by up to 54%. Re-growth assays showed that the improved survival rates of LNCaP/T cells after radiation did not change after 3 days, remaining constant over 3 weeks. CONCLUSIONS: These results identify clusterin as a promoter of cell survival that may help mediate resistance to radiation-induced apoptosis. Furthermore, clusterin overexpression seems to provide an extended protection against radiation-induced cell cycle arrest and apoptosis.

Acquisition of resistance to Fas-mediated apoptosis by overexpression of clusterin in human renal-cell carcinoma cells.

Recent studies have shown the antiapoptotic activity of clusterin against a wide variety of stimuli; however, the functional role of clusterin in Fas-mediated apoptosis has not been well characterized. We transfected the clusterin cDNA into human renal-cell carcinoma (RCC) ACHN cells that scarcely express clusterin protein in order to examine whether overexpression of clusterin inhibits the Fas-mediated signal pathway for apoptotic cell death. No significant difference was observed in the in vitro cell growth rates between the clusterin-transfected cell line (ACHN/CL) and the vector-only-transfected control cell line (ACHN/C), whereas the colony-forming efficiency in soft agar of ACHN/CL was significantly higher than that of ACHAN/C. The anti-Fas monoclonal antibody CH11 induced apoptosis in ACHAN/C cells in a dose-dependent manner; however, the growth-inhibitory effect of CH11 on ACHN/CL cells was markedly suppressed, with corresponding increases in p53 expression and decrease in the fraction of cells in the sub-G(1) phase of the cell cycle. Furthermore, the cytotoxic effect of CH11 on ACHN/CL cells was augmented by treatment with interferon-gamma, but a corresponding effect on ACHN/C cells was not observed. These findings suggest that overexpression of clusterin may contribute to a phenotype resistant to Fas-mediated apoptosis, and that if interferon-gamma treatment is added according to the clusterin expression level, Fas-mediated therapy could be a novel approach to RCC.

Chemosensitization of human renal cell cancer using antisense oligonucleotides targeting the antiapoptotic gene clusterin.

BACKGROUND: Renal cell cancer (RCC) is a chemoresistant disease with no active chemotherapeutic agent achieving objective response rates higher than 15%. Clusterin is a cell survival gene that increases in human renal tubular epithelial cells after various states of injury and disease. Downregulation of clusterin, using antisense oligonucleotides (ASO), has recently been shown to increase chemosensitivity in several prostate cancer models. The objectives in this study were to evaluate clusterin expression levels in human RCC and normal kidney tissue, and to test whether clusterin ASO could also enhance chemosensitivity in human RCC Caki-2 cells both in vitro and in vivo. METHODS: Immunohistochemical staining was used to characterize clusterin expression in 67 RCC and normal kidney tissues obtained from radical nephrectomy specimens. Northern blot analysis was used to assess changes in clusterin mRNA expression after ASO and paclitaxel treatment. The effects of combined clusterin ASO and paclitaxel treatment on Caki-2 cell growth was examined using an MTT assay. Athymic mice bearing Caki-2 tumors were treated with clusterin ASO alone, clusterin ASO plus paclitaxel, and mismatch control oligonucleotides plus paclitaxel, over a period of 28 days with measurement of tumor volumes once weekly over 8 weeks. RESULTS: Immunohistochemistry of normal and malignant kidney tissue sections of 67 patients demonstrated positive clusterin staining for almost all RCC (98%) and an overexpression, compared to normal tissue, in a majority of RCC (69%). Clusterin ASO, but not mismatch control oligonucleotides, decreased clusterin mRNA expression in Caki-2 cells in a dose-dependent and sequence-specific manner. Pretreatment of Caki-2 cells with clusterin ASO significantly enhanced chemosensitivity to paclitaxel in vitro. Characteristic apoptotic DNA laddering was observed after combined treatment with ASO plus paclitaxel, but not with either agent alone. In vivo administration of clusterin ASO plus paclitaxel acted synergistically to increase apoptosis and significantly delay Caki-2 tumor growth, compared to mismatch control oligonucleotide plus paclitaxel. In addition, TUNEL staining revealed increased apoptotic cells in tumors treated with clusterin ASO plus paclitaxel compared to treatment with either clusterin ASO or paclitaxel alone. CONCLUSION: These findings confirm that the use of clusterin ASO may be a feasible strategy to enhance chemosensitivity for patients with advanced RCC.

Antitumor activity of antisense clusterin oligonucleotides is improved in vitro and in vivo by incorporation of 2'-O-(2-methoxy)ethyl chemistry.

Phosphorothioate (P=S) antisense oligonucleotides (ASO) targeting the cell survival gene clusterin synergistically enhance castration- and chemotherapy-induced apoptosis in prostate cancer xenografts. This study compares efficacy, tissue half-lives, and toxicity of P=S clusterin ASO to third-generation backbone 2'-O-(2-methoxy)ethyl (2'MOE) ribose-modified clusterin ASO. Northern analysis quantified changes in clusterin mRNA levels in human PC-3 cells and tumors. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay measured effects of combined clusterin ASO plus paclitaxel on PC-3 cell growth. Athymic mice bearing PC-3 tumors were treated with paclitaxel plus either P=S clusterin ASO, 2'-MOE clusterin ASO, or mismatch control oligonucleotides for 28 days. Weekly body weights and serum parameters were measured to assess toxicity. Tissue half-life of P=S and 2'-MOE ASO in PC-3 tumors was assessed using capillary gel electrophoresis (CGE). Both 2'-MOE and P=S ASO decreased clusterin mRNA levels in a dose-dependent and sequence-specific manner. 2'-MOE ASO more potently suppressed clusterin mRNA (80 versus 40% at 500 nM) compared with P=S ASO. IC(50) of paclitaxel was equally reduced (50--75%) by both compounds. In vivo tissue half-life was significantly longer for 2'-MOE-modified ASO than for P=S ASO (5 versus 0.5 days). Using CGE, >90% of detected 2'-MOE ASO in tumor tissue was full length. Weekly administration of 2'-MOE clusterin ASO was equivalent to daily P=S clusterin ASO in enhancing paclitaxel efficacy in vivo. 2'-MOE-modified ASO potently suppressed clusterin expression and prolonged tissue half-lives with no additional side effects. These results support the use of 2'-MOE-modified ASO over conventional P=S ASO by potentially increasing potency and allowing longer dosing intervals in clinical trials.

Use of antisense oligonucleotides targeting the antiapoptotic gene, clusterin/testosterone-repressed prostate message 2, to enhance androgen sensitivity and chemosensitivity in prostate cancer.

BACKGROUND: Androgen resistance develops, in part, from upregulation of antiapoptotic genes after androgen withdrawal. Identification and targeting of genes mediating androgen-independent (AI) progression may lead to development of novel therapies that delay hormone-refractory prostate cancer. Clusterin is a cell survival gene, that increases after androgen ablation. Here, we review clusterin's functional role in apoptosis and the ability of antisense oligonucleotides (ASOs) against clusterin to enhance apoptosis in prostate cancer xenograft models. RESULTS: Immunostaining of radical prostatectomy specimens confirm that clusterin is highly expressed in 80% prostate cancer cells after neoadjuvant hormone therapy, but is low or absent (<20%) in untreated specimens. Clusterin levels increase >10 fold in regressing Shionogi tumors after castration. Pretreatment of mice bearing androgen-dependent Shionogi tumors with calcium antagonists inhibited castration-induced apoptosis, tumor regression, and clusterin gene upregulation, illustrating that clusterin is an apoptosis-associated gene and not an androgen-repressed gene. Clusterin ASOs reduced clusterin levels in a dose-dependent and sequence-specific manner. Adjuvant treatment with murine clusterin ASOs after castration of mice bearing Shionogi tumors decreased clusterin levels by 70% and resulted in earlier onset and more rapid apoptotic tumor regression, with significant delay in recurrence of AI tumors. Species-specific clusterin ASOs also increased the cytotoxic effects of paclitaxel, reducing the 50% inhibitory concentration (IC(50)) of PC-3 and Shionogi cells by 75% to 90%. Although clusterin ASOs had no effect on the growth of established AI Shionogi or PC-3 tumors, clusterin ASOs synergistically enhanced paclitaxel-induced tumor regression in both Shionogi and PC-3 models. CONCLUSIONS: Collectively, these data identify clusterin as an antiapoptosis protein, upregulated in an adaptive cell-survival manner by androgen ablation and chemotherapy, which confers resistance to various cell-death triggers. Inhibition of clusterin upregulation using clusterin ASOs can enhance cell death after treatment with androgen ablation and chemotherapy.

Synergistic chemosensitization and inhibition of progression to androgen independence by antisense Bcl-2 oligodeoxynucleotide and paclitaxel in the LNCaP prostate tumor model.

Bcl-2 expression is up-regulated in prostate cancer cells after androgen ablation and associated with development of androgen independence and chemoresistance. We recently reported that antisense Bcl-2 oligodeoxynucleotides (ODNs) delay progression to androgen independence in the androgen-dependent (AD) human LNCaP prostate tumor model. The objectives in this study were to determine whether antisense human Bcl-2 ODN enhances chemosensitivity of paclitaxel and whether combined antisense Bcl-2 ODN and paclitaxel further delays time to androgen-independent (AI) progression in the LNCaP tumor model. Semi-quantitative reverse transcriptast-polymerase chain reaction revealed that treatment of LNCaP cells with antisense Bcl-2 ODN decreased Bcl-2 expression in a dose-dependent and sequence-specific manner, whereas Bcl-2 expression was not affected by paclitaxel treatment. Antisense Bcl-2 ODN treatment significantly enhanced paclitaxel chemosensitivity in vitro, reducing cell viability after treatment with 1 nM paclitaxel from 76% to 42%. Characteristic apoptotic DNA laddering was demonstrated after combined treatment with 500 nM antisense Bcl-2 ODN and 1 nM paclitaxel but not with either agent alone. Adjuvant in vivo administration of combined antisense Bcl-2 and polymeric micellar paclitaxel after castration resulted in a significant delay of emergence of AI recurrent LNCaP tumors compared with either agent alone. By 15 weeks post castration, tumor volume in mice treated with antisense Bcl-2 ODN alone or mismatch control ODN plus paclitaxel was >3-fold higher than in mice treated with combined antisense Bcl-2 ODN and paclitaxel. Mean serum prostate-specific antigen levels returned to or were above precastration levels by 11 weeks post castration in mice treated with antisense Bcl-2 ODN alone or mismatch control ODN plus paclitaxel but remained 90% below the pre-castration level in mice treated with combined antisense Bcl-2 ODN and paclitaxel. These findings identify combined antisense Bcl-2 and paclitaxel as a potentially new therapeutic strategy for advanced prostate cancer by enhancing paclitaxel chemosensitivity and delaying progression of hormone-refractory prostate cancer.

Chromosomal imbalances in noninvasive papillary bladder neoplasms (pTa).

Almost 70% of urinary bladder neoplasms present as low-grade papillary noninvasive tumors (stage pTa). To determine which genomic alterations can occur in pTa tumors of different grades and to evaluate the prognostic significance of chromosomal imbalances, we analyzed 113 pTa tumors (40 grade 1, 55 grade 2, 18 grade 3) by comparative genomic hybridization. pTaG1 (1.9 +/- 2.0) and pTaG2 (3.1 +/- 2.9) tumors had only few genomic alterations with 9q- (44%), 9p- (36%), and -Y (21%) being most prevalent. Neither the total number of aberrations nor any individual alteration was linked to the risk of recurrence in 95 pTaG1/G2 tumors with clinical follow-up information. pTaG3 tumors were characterized by a high number of alterations (7.7 +/- 4.5; P < 0.0001 for G3 versus G2). Several chromosomal imbalances that have previously been reported to be typical for invasive bladder neoplasms were significantly more frequent in pTaG3 than in pTaG2 tumors, including 2q-, 5p+, 5q-, 6q-, 8p-, 10q-, 18q-, and 20q+. A malfunction of genes at these loci may contribute to the development of high-grade urothelial neoplasias. However, there is no evidence for a direct role of these alterations for development of invasive tumor growth.

Cyclin D1 overexpression lacks prognostic significance in superficial urinary bladder cancer.

The biological behaviour of urinary bladder neoplasms cannot be adequately predicted by histological criteria alone. Cyclin D1 is a cell-cycle regulating protein known to be overexpressed in a proportion of bladder carcinomas. To evaluate the prognostic significance of cyclin D1 expression and its relationship with tumour phenotype, 392 bladder carcinomas were analysed by immunohistochemistry. Clinical follow-up information was available in 337 patients with superficial bladder tumours (stages pTa/pT1). Cyclin D1 positivity was seen in 176 of 392 carcinomas. Cyclin D1 overexpression was strongly linked to papillary tumour growth, low stage, and low histological grade (p<0.005 each). Multivariate analysis showed that papillary tumour growth was the only parameter which was independently linked to cyclin D1 positivity. There was no significant difference in proliferative activity (Ki67 labelling index) between cyclin D1-negative and -positive tumours. Cyclin D1 positivity was not linked to the risk of recurrence or tumour progression, either in pTa or in pT1 carcinomas. It is concluded that cyclin D1 positivity distinguishes a large subgroup of papillary bladder tumours, but there is no evidence of prognostic significance for increased cyclin D1 expression.

Polysomies but not Y chromosome losses have prognostic significance in pTa/pT1 urinary bladder cancer.

A disturbed cellular DNA content is of potential diagnostic and prognostic relevance in urinary bladder cancer. To evaluate the prognostic significance of individual chromosomal aberrations in superficial bladder cancer, specimens of 105 tumors (67 pTa, 38 pT1) were examined by fluorescence in situ hybridization (FISH). FISH allows quantitation of chromosomes on a cell by cell level. Centromere probes for the chromosomes Y, 1, and 17 were used. There was a strong association between polysomies of the chromosomes 1 (found in 46% of tumors) and 17 (40% of tumors, P < .0001). Polysomies (1 and 17) were significantly more frequent in pT1 than in pTa tumors (P < .0001 each). In pTa tumors, polysomies of both chromosomes were linked to a high risk of recurrences; polysomy 17 was associated with an increased risk of progression (P < .05 each). There was no significant association between polysomies and an unfavorable prognosis in pT1 carcinomas. Previous studies had suggested a prognostic role of Y losses in bladder cancer. However, Y losses were not linked to recurrences or tumor progression in pTa or pT1 tumors of 67 male patients. These data show that marked genetic differences exist between pTa and pT1 carcinomas. They also indicate that polysomies of different chromosomes may have prognostic relevance in pTa urinary bladder cancer.